Process of extracting biotin



Patented Apr. 24, 1945 PROCESS OF EXTRACTING BIOTIN Joseph V. Karabirios, Columbus,.and Elmer R. Eckhardt and Wilbur I. Patterson, Chagrin Falls, Ohio, asslgnors to S. M. A. Corporation, Chicago, Ill., a corporation of New Jersey No Drawing. Application July 13,1943, Serial No. 494,588

4 Claims.

This invention relates to the process of preparing a vitamin preparation having biotin activity and using as starting materials plant sources such as the fermentation residues from molasses and corn or other grains.

Heretofore preparations having biotin activity have used as process starting materials various animal source products such as the liver and other animal organs.

The isolation of biotin by any of the known processes is a long and expensive process not well suited to the large scale production of biotin or preparations having biotin activity. Examples of this are the isolation of biotin from egg yolk by Kogl and Tonnis (Z. Physiol. Chem., 242, 43 (1936) which illustrates the difliculties encountered. The process described by du Vigneaud, Hoffman, Melville and Gyorgy (J;

Biol. Chem., 140,643, (1941) for the isolation of biotin from liver, while being a simpler process than the one from egg yolk still leaves much to be desired from cost and manufacturing difflculties standpoint.

We have discovered that a preparation having biotin activity may be selectively extracted by the use of certain organic solvents from fermentation residues of molasses and corn or other grain thereby simplifying to a great degree the hitherto known processes and using a, starting material low incest and readily obtainable.

Biotin (vitamin H) has been recognized as a nutritional factor having the power to prevent or cure the dietary deficiency produced by a diet containing a high percentage of uncooked egg white.

This present application is not limited to the biotin molecule with the same properties as those described by the investigators listed above but is directed to a vitamin preparation having biotin activity when assayed by the methods employed by us. The invention is based upon our discovery that the plant materials containing compositions having biotin activity, when extracted with suitable organic solvents such as ethylene dichloride, esters, (ethyl acetate), ketones (acetone) and alcohols such as ethyl, isobutyl, ethanol and methanol, transferred the biotin activity to the solvent used. Such solvents, however, are not used in the sense of ordinary chemical extraction but must be solvents in which water is appreciably soluble since water plays an important role in the extraction process, either as a control concentration for the solvent or where such water appears as moisture in the starting material.

We have found an excellent starting material for practicing our invention to be the dried or partially dried molasses fermentation residue product sold commercially under the trademarks Curbay BG" and-BY Feed and while we do not desire to limit ourselves to the particular product of one manufacturer, we hav found these two products suitable.

For the .purpose of illustration, several examples of extraction by individual solvents are hereinafter set forth and the results of assays of the found end-product appended thereafter. While the products sold under the above trade-marks are usually known to the trade as spray dried molasses fermentation residues, we do not desire to limit ourselves to the use of spray dried starting materials, since any solid material containing suitable amounts of biotin and not containing excessive amounts of moisture can be used with equal ease a the starting materials as will hereinafter appear.

Example 1.--0ne hundred grams of spray dried molasses fermentation residue having an assay of about three micrograms of biotin per gram were refluxed in a suitable apparatus for one and one-half hours with 200 cc. of ethyl acetate saturated with water. The solid is then removed by filtration. An assay of the filtrate shows it to contain biotin activity equivalent to one hundred and six micrograms of biotin and the residue obtained by evaporation weighs approximately 400 milligrams. A 34% yield of material having biotin activity is' thus realized with an -fold concentration of such activity.

Example 2.-- lbs. of spray dried molasses fermentation residue which will assay about, 2.5 micrograms of biotin per gram are placed in a cylindrical vessel of suitable material having a cone-shaped bottom and a delivery tube. 45

gallons of acetone previously treated to contain- 5% of water are allowed to flow to the vessel from a container at such a rate that the solid in the vessel remains immersed below the level of the liquid. Approximately 30 gallons of percolate are collected and the acetone removed therefrom by distillation. The recovered acetone may be used in subsequent processes after the water content, thereof has been readjusted to 5%. The residue secured by the abov process contains 40 milligrams of biotin activity in 1200 grams of solids, thereby representing a 15- fold concentration. No such amount of biotin activity or comparable concentration can be secured unless the water content of the acetone is in the neighborhood of 5%.

Example 3.One pound of molasses fermentation residue is percolated with one liter of commercial methanol by the method described Solvents other than those specifically set forth may be used it they meet the requirements tor moisture content and have thedesired extracin Example 2. In this particular example it tive properties. should be noted that the starting material is I What we claim is: not spray dried, but must contain an amount 1- The process of preparin 8 83 1 havins of moisture not appreciably greater than 5% in biotin-like activity which pr s s extractin 8 order that a proper yield of biotin activity in molasses fermentation residue with an or ni the extract can be obtained. By filtration a solvent selected from the gr p Consisting of acelight brown filtrate is obtained which assays 530 10 one. ethyl alcohol, methyl alcohol, ethyl acetate, micrograms or 53% and has a biotin potency of a d thylene dichloride, containin water in a 13 micrograms per gram of total solids. Small a ount not exceeding about 5%, a d ev Erample 4.-50 grams of molasses fermentacrating oil the solvent and water from the retion residue which will assay 3.0 micrograms of Suiting extract to Obtain a residue having a t bioti per gram are extracted by vigorous m. in fold concentration in excess of about five timesin rin in b ili ab l t ethyl alcohol f one biotin-like activity over that of said molasses ierhour and then filtered. The filtrate assays 45 mutation residuemicrograms of biotin for a 30% yield with total The P ss of preparin 8- t l ha solids of 2.6 grams. Here against the moisture m8 biotin-like ahtivity which 1 8 5 e t tcontent of the starting material is important. ing a molasses fermentation residue with acetone Example 5 5o grams of spra dried molasses containing about 5% water, and evaporating fermentation residue which will assay 3.0 micro- Off the acetone n Water from the resulting e grams of b t per gram are extracted by tract to obtain a residue having a multi-fold conomus stirring in a boiling mixture of equal parts centration of about 13 times in biotin-like activity of ethyl 1 1 1 and ethyl acetate containing 5% over that of said molasses fermentation residue. of water. Filtration from the insoluble material The Pr ss of preparing a material having yields a Solution containing biotin activity biotin-like activity which comprises extracting a equivalent t 68 micrograms of bi t in 5 molasses fermentation residue with methyl alcograms of tot solids indicating a 45% yield, hol containing about 5% water, and evaporating Example 6.100 grams of spray drie m l oil the methyl alcohol and water from the resultfermentation residue which will assay about 5 ns extract to obtain a residue havin a micrograms of biotin activity per gram are stirre fold concentration of about five times in biotini'or three hours on a steam bath with 250 cc. of like activity over that of said molasses fermentaethylene dichloride to which has been added 4 cc. n r eof water which represents the approximate satu- 4. The process of pr p ri a ma ri having rated solubility. A light yellow filtrate results biotin-like activity whic Comprises extracting 8 which ns 430 c ograms of material havmolasses fermentation residue with a mixture ing biotin activity in 400 mg. of solids which reconsisting of equal parts of ethyl acetate and suited after the solvent hadbeen removed in ethyl alcohol and containing about 5% water, vacuo. This represents a 240-told purification 40 and evaporating oil the ethyl acetate, ethyl alcoand a nearly theoretical recovery. If the water hol, and water to obtain a residue having a multiis not added to the ethylene dichloride less than fold concentration of about five times in biotinr c of the material having the biotin activity can like activity over that of said molasses fermentabe extracted. tion residue.

Table I Biotin Purity some Method 3 g grams per gram Acetone (5?, water) Percolation 100 lbs- 38,600 32 18 fold. Methanol commercial) do 400grams. 530 13.8 5.6 fold. Ethanol (Solox") Hot extraction BOgramlL. 17.2 Hold. Isobutyl alcohol (5% water) grams" 72 i6. 7 7 fold. Ethyl acetate and ethyl alcohol (-50) -.do 50 grams. 68 12.4 Mold. a zimm (2% water) .do grams. 106 28.0 Shield. Ethylene dichloride (1% water) do 100 grams. 480 1,3 240 told.

Similar extractions from fermentation residues JOSEPH of corn and other grains can be secured by utilization of related processes.

V. KARABINOS. ELDER R. ECKHARDT. 

